Transcriptome analysis of granulosa cells after conventional vs long FSH-induced superstimulation in cattle.

BACKGROUND: Prolongation of superstimulatory treatment appears to be associated with a greater superovulatory response and with greater oocyte maturation in cattle. A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25 mg FSH im at 12-h intervals; n = 6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 h later. Granulosa cells were harvested 24 h after LH treatment. RESULTS: The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) supported the microarrays data. Multigene bioinformatic analysis indicates that markers of fertility and follicle maturity were up-regulated in the long FSH group. CONCLUSION: Using the large gene expression dataset generated by the genomic analysis and our previous associated with the growth phase and gene expression changes post LH, we can conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle.

Ovulation-inducing factor (OIF/NGF) in seminal plasma: a review and update.

The ovulation-inducing effect of seminal plasma was first reported in Bactrian camels over 30 years ago, and the entity responsible was dubbed 'ovulation-inducing factor' (OIF). More recent studies, primarily in llamas and alpacas, characterized the biological and chemical properties of OIF and ultimately identified it as ßNGF. This recent discovery has allowed a convergence of knowledge previously separated by discipline and by mechanism; that is, neurobiology and reproductive biology, and autocrine/paracrine vs endocrine. To preserve this link, we have referred to the seminal factor as OIF/NGF. As a highly conserved protein, the implications of discoveries related to OIF/NGF in reproductive tissues extend beyond the camelid species, and results of recent studies show that the presence and function of OIF/NGF in seminal plasma are conserved among species considered to be induced ovulators as well as those considered to be spontaneous ovulators. The abundance of OIF/NGF in seminal plasma and the effects of seminal plasma on ovarian function strongly support the idea of an endocrine mode of action (i.e. systemic distribution with distant target tissues). This review is intended to provide an update on the progress in our understanding of the nature of OIF/NGF in seminal plasma and its effects on reproductive function in the female, including the effects of dose and route of administration, evidence for ovarian effects in other species, tissue sources of OIF/NGF and early findings related to the mechanism of action of OIF.

Organelle reorganization in bovine oocytes during dominant follicle growth and regression.

BACKGROUND: We tested the hypothesis that organelles in bovine oocytes undergo changes in number and spatial distribution in a manner specific for phase of follicle development. METHODS: Cumulus-oocyte-complexes were collected from Hereford heifers by ultrasound-guided follicle aspiration from dominant follicles in the growing phase (n = 5; Day 0 = ovulation), static phase (n = 5), regressing phase (n = 7) of Wave 1 and from preovulatory follicles (n = 5). Oocytes were processed and transmission electron micrographs of ooplasm representing peripheral, perinuclear and central regions were evaluated using standard stereological methods. RESULTS: The number of mitochondria and volume occupied by lipid droplets was higher (P < 0.03) in oocytes from regressing follicles (193.0 ± 10.4/1000 µm(3) and 3.5 ± 0.7 %) than growing and preovulatory stages (118.7 ± 14.4/1000 µm(3) and 1.1 ± 0.3 %; 150.5 ± 28.7/1000 µm(3) and 1.6 ± 0.2 %, respectively). Oocytes from growing, static and preovulatory follicles had >70 % mitochondria in the peripheral regions whereas oocytes from regressing follicles had an even distribution. Oocytes from growing follicles had more lipid droplets in peripheral region than in central region (86.9 vs. 13.1 %). Percent surface area of mitochondria in contact with lipid droplets increased from growing (2.3 %) to static, regressing or preovulatory follicle stage (8.9, 6.1 and 6.2 %). The amount, size and distribution of other organelles did not differ among phases (P > 0.11). CONCLUSIONS: Our hypothesis was supported in that mitochondrial number increased and translocation occurred from a peripheral to an even distribution as follicles entered the regressing phase. In addition, lipid droplets underwent spatial reorganization from a peripheral to an even distribution during the growing phase and mitochondria-lipid contact area increased with follicle maturation.

Effect of vehicle and route of administration of letrozole on ovarian function in a bovine model.

The objective of this study was to determine the effects of vehicle and route of administration of letrozole on ovarian function in sexually mature beef heifers. On Day 3 (Day 0=ovulation), heifers were assigned randomly to four treatment groups and given 1mgkg(-1) letrozole intravenously (iv, n=10) or intramuscularly (im, n=10) or given a placebo iv (control iv, n=5) or im (control im, n=5). The interwave interval was longer in heifers treated with letrozole im than in im and iv controls (11.7±0.30 vs 9.5±0.50 and 10±0.43, respectively; P<0.05). Corpus luteum diameter profiles and plasma progesterone concentrations were greater (P<0.03 and P<0.05, respectively) in heifers treated with letrozole im compared with control im. Plasma oestradiol concentrations were lower in both letrozole-treated groups compared with controls (P≤0.03). Plasma LH concentrations tended to be elevated at the time of wave emergence in heifers treated with letrozole im compared with other groups (group-by-day interaction, P=0.06) and plasma FSH concentrations tended to be greater (P<0.09) in heifers treated with letrozole by either route compared with a single control group. We conclude that intramuscular administration of letrozole in oil is a feasible route and vehicle for the development of a letrozole-based treatment protocol for herd synchronisation in cattle.

Predictive characteristics of male fertility in alpacas with special reference to seminal NGF.

Recent studies document the LH-releasing pathway of nerve growth factor (NGF) in male camelids and that the LH response to seminal NGF is associated with elevated plasma testosterone concentration. Results provide rationale for the hypothesis that NGF in semen is associated with male fertility. In Experiment 1, the association between the amount of NGF in the ejaculate and characteristics of the male reproductive system was examined in alpacas. The concentration of NGF was measured by radioimmunoassay in semen samples collected from male alpacas (n = 47) and correlated with sperm morphology and motility, and measurements of the male reproductive anatomy. Most ejaculates had NGF concentrations that, based on previous studies, triggered ovulation in female camelids, however, we only found a positive correlation between NGF concentration with sperm concentration, thread formation and total NGF, and a negative correlation with pH. In Experiment 2, a retrospective analysis was carried out to determine if breeding performance during the previous season was related to recent concentrations of seminal NGF in male alpacas (n = 22). Birth rates tended to be correlated with sperm concentration and total amount of NGF in the ejaculate (P = 0.09). Experiment 3 was a prospective study to determine the relationship between seminal NGF (n = 8 male alpacas) and ovulation and pregnancy rates in a breeding trial. No association was detected between seminal NGF concentration and ovulation rate, pregnancy rate, or LH response in the female. We conclude that among the breeding males used in our study, the abundance of seminal NGF was correlated with sperm concentration and thread formation, however, it was not predictive of male fertility in alpacas. Examination of males not previously selected as breeding stock may be expected to include a broader range of seminal NGF and provide a more comprehensive understanding of the relationship between seminal NGF and male fertility.

Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle.

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

Ultrasound biomicroscopy: a non-invasive approach for in vivo evaluation of oocytes and small antral follicles in mammals.

The use of ultrasonography has changed our understanding of the ovarian function in live animals. However, most of the studies that have used ultrasonography to image the ovary have provided data only of structures >1mm in diameter. The recent availability of high-resolution ultrasound technology with high-frequency transducers (25-70 MHz), offers the potential to examine the developmental dynamics of small antral follicles and the cumulus-oocyte complex (COC) in vivo. In this review we provide data from a series of studies performed by Veterinary Biomedical Sciences Laboratory describing the advantages and disadvantages, as well as image characteristics, of ultrasound biomicroscopy (UBM) to study ovarian biology in mammals. Data and images of small ovarian structures in rabbits, cattle, mice and humans are shown. The UBM technique allowed visualisation of small antral follicles ranging in size from 300 to 700 µm in all species examined, as well as COC within follicles in rabbits, cattle and humans. Furthermore, UBM permitted clear distinction of the follicular wall from the surrounding ovarian stroma in cattle and humans. At present, the limited depth of penetration of UBM restricts the use of this technique to an experimental setting. In that regard, further studies using UBM will probably result in a greater understanding of the pattern and control of early antral folliculogenesis and oogenesis.

Effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle.

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n=14), FSH starvation (n=13) and long FSH (n=13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P=0.001), and ova and/or embryos (P<0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate.

Development and Biodistribution of a Nerve Growth Factor Radioactive Conjugate for PET Imaging.

PURPOSE: The purpose of these studies was to develop a nerve growth factor (NGF) radiometal-chelator conjugate to determine the biodistribution and brain uptake of NGF by positron emission tomography/computerized tomography (PET-CT). PROCEDURES: Purified NGF from llama seminal plasma was conjugated with FITC, and the chelator NOTA or DFO. NGF conjugates were evaluated for bioactivity. NOTA- and DFO-conjugated NGF were radiolabeled with gallium-68 or zirconium-89 ([68 Ga]GaCl3, half-life = 68 min; [89Zr]Zr(oxalate)4, half-life = 3.3 days). [89Zr]Zr-NGF was evaluated for biodistribution (0.5, 1, or 24 h), PET imaging (60 min), and brain autoradiography in mice. RESULTS: Cell-based in vitro assays confirmed that the NGF conjugates maintained NGF receptor-binding and biological activity. Zirconium-89 and gallium-68 radiolabeling showed a high efficiency; however, only[89Zr]Zr-NGF was stable in vitro. Biodistribution studies showed that, as with most small proteins < 70 kDa, [89Zr]Zr-NGF uptake was predominantly in the kidney and was cleared rapidly with almost complete elimination of NGF at 24 h. Dynamic PET imaging from 0-60 min showed a similar pattern to ex vivo biodistribution with some transient liver uptake. Interestingly, although absolute brain uptake was very low, at 24 h after treatment, cerebral cortex uptake was higher than any other brain area examined and blood. CONCLUSIONS: We conclude that conjugation of DFO to NGF through a thiourea linkage allows effective radiolabeling with zirconium-89 while maintaining NGF bioactivity. Following intravenous administration, the radiolabeled NGF targets non-neuronal tissues (e.g., kidney, liver), and although absolute brain uptake was very low, the brain uptake that was observed was restricted to the cortex.

Predictors of the ovarian superstimulatory response and oocyte collection in prepubertal heifers.

The objectives were to investigate the relationships between antral follicle counts and plasma AMH and FSH at the time of follicular wave emergence in prepubertal calves, and to determine the effects of age and duration of gonadotropin treatment on the ovarian superstimulatory response in pre- and post-pubertal heifers. Hereford crossbred prepubertal (Replicate 1 and 2, n = 20) and post-pubertal heifers (Replicates 1, n = 8; Replicate 2, n = 8) were assigned randomly to 2 treatment groups and given FSH for either 4 or 7 d (25 mg pFSH im at 12-h intervals). Prepubertal heifers were first treated at 4 mo and again at 7 mo of age. Blood samples were collected immediately before the first FSH administration, that was initiated 36 h after follicular ablation. An LH treatment (12.5 mg im) was given 12 h after the last FSH injection. Follicular fluid and cumulus-oocyte complexes (COC) were collected 24 h after LH treatment. At wave emergence, the number of follicles ≥1 mm (AFC, 31.1 ± 4.0 vs 16.2 ± 1.8; P < 0.001) and the plasma concentrations of AMH (606.4 ± 90.5 vs 279.6 ± 28.3 pg/mL; P = 0.001) were higher at 4 than at 7 mo of age, while plasma FSH concentrations did not differ between ages. At oocyte collection, a higher number of follicles ≥6 mm were observed in prepubertal calves at 4 mo of age and post-pubertal heifers than in calves at 7 mo of age (32.4 ± 5.4 and 22.0 ± 2.3 vs 14.9 ± 2.0, respectively; P = 0.003). Intrafollicular concentrations of estradiol were lower (23.7 ± 4.5 vs 144.0 ± 29.5 ng/mL; P < 0.0001) and of progesterone tended to be higher (217.5 ± 29.3 vs 157.0 ± 33.9 ng/mL; P = 0.07) in the 7- than in the 4-d groups. A greater number of COC was collected from calves at 4 mo of age and heifers than the 7-mo-old calves (13.4 ± 2.6 and 6.0 ± 1.0 vs 5.8 ± 1.1, respectively; P = 0.008). Overall, the 7-d FSH treatment tended to result in a greater proportion of expanded COC than the 4-d treatment in calves (50.1 ± 7.7 vs 31.9 ± 6.8%; P = 0.07). In summary, there was a positive relationship between AFC and plasma AMH concentrations at the time of wave emergence. A higher AFC was observed in calves at 4- than 7-mo of age, which resulted in greater ovarian response to gonadotropin treatment. Following an exogenous LH stimulus, COC maturation rates were greater in the 7-d than in the 4-d FSH treatment groups, resulting in collection of a higher proportion of fully expanded COC.

Evidence for the conservation of biological activity of ovulation-inducing factor in seminal plasma.

An ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas (induced ovulators) and cattle (spontaneous ovulators) suggests that OIF is a conserved constituent of seminal plasma among mammals. In this study, three experiments were designed to determine the biological effects of OIF in different species. In experiment 1, superstimulated prepubertal female CD-1 mice (n=36 per group) were given a single 0.1 ml i.p. dose of 1) phosphate-buffered saline (PBS), 2) 5 µg gonadotropin-releasing hormone (GNRH), 3) 5 IU hCG, or 4) llama seminal plasma. The proportion of mice that ovulated was similar among groups treated with GNRH, hCG, or seminal plasma, and all were higher than the saline-treated group (P<0.001). In experiment 2, female llamas (n=8 or 9 per group) were intramuscularly treated with 1) 2 ml PBS, 2) 1 ml diluted llama seminal plasma, 3) 3 ml equine seminal plasma, or 4) 3 ml porcine seminal plasma. Experiment 3 was the same as experiment 2 except that the dose of equine and porcine seminal plasma was increased to 8 and 10 ml respectively. All llamas that were treated with llama seminal plasma ovulated and none that were treated with saline ovulated (P<0.0001). The proportion of llamas that ovulated in response to equine and porcine seminal plasma was intermediate. We conclude that the mechanism for the biological response to OIF is present in prepubertal CD-1 mice and that OIF is present in equine and porcine seminal plasma.

Comparison of two intravaginal progesterone-releasing devices in shortened-timed artificial insemination protocols in beef cattle.

Commercially available intravaginal progesterone (P4) devices differ in shape, surface area and P4 load, which may affect the resulting pregnancy per AI (P/AI) following timed-AI (TAI). The objective of this study was to compare two intravaginal P4 devices on estrus rate, follicular dynamics and P/AI in beef cattle subjected to shortened-TAI protocols. In Expt. 1, nulliparous heifers were randomly assigned to a P4-releasing intravaginal device (PRID-Delta, 1.55 g P4) or a controlled internal drug release (CIDR, 1.38 g P4) at the initiation of a J-synch protocol. Heifers that displayed estrus 72 h following device removal were TAI, or if not in estrus given GnRH at 72 h and TAI at 90 h. In Expt. 2, nulliparous heifers and non-suckling cows were randomly assigned to either PRID or CIDR groups and either 1 or 2 mg of estradiol benzoate (EB) at initiation of a J-synch protocol. All cattle were TAI concurrent with GnRH 72 h after device removal. In Expt. 3, nulliparous heifers and suckling cows were randomly assigned to either PRID or CIDR groups and initiated a 5-d Cosynch protocol, with TAI concurrent with GnRH 72 h following device removal. In each experiment, cattle received estrus detection patches at device removal, which were then scored from 0 to 3 based on color change between initial application and TAI; 0 = unchanged, 1 = ≤50% change, 2 = >50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. Transrectal ultrasonography was used to determine cyclicity, diagnose pregnancy in all experiments, and the size of the ovulatory follicle in Expt. 3. In Expt. 1, the estrus rate was greater (72.0% vs. 61.0%; P = 0.04) in the PRID compared to the CIDR group. In Expt. 2, a parity by EB dose interaction (P = 0.02) was attributed to an increased estrus rate (52.8% vs. 41.4%; P = 0.05) in heifers given 1 vs. 2 mg EB. In Expt. 3, there was no difference in the ovulatory follicle diameter at device removal (P = 0.22) or TAI (P = 0.28) between P4 groups. Treatment with a PRID tended (P = 0.10) to increase the P/AI in cows compared to a CIDR (73.5% vs. 61.0%). In all experiments combined, the overall P/AI tended to increase (55.2% vs. 51.0%; P = 0.08) and P/AI in cattle exhibiting estrus increased (64.4% vs. 59.7%; P = 0.02) in cattle given a PRID compared to those given a CIDR, respectively. In summary, the type of intravaginal P4 device affected estrus response and P/AI following TAI in beef cows.

Research and development of a silicone letrozole-releasing device to control reproduction in cattle.

Two experiments were conducted to test the effectiveness of a silicone matrix as an intravaginal drug delivery device for letrozole, an aromatase inhibitor used for synchronization protocols in cattle. A wax dip-coat formulation of the intravaginal device used in previous studies was effective in releasing letrozole but was cumbersome to manufacture and deploy, resulting in unwanted variation in drug delivery and circulating concentrations of letrozole. In Experiment 1, a 3 × 3 design was used to test the release kinetics of letrozole from silicone in vitro. Silicone was mixed with 3 different letrozole drug loads (5%, 10%, 15%) and 3 different mineral oil loads (5%, 10%, 15%), and letrozole release into 62.5% ethanol was compared with the wax dip-coat formulation (positive control) by UV spectrophotometry. Letrozole was released from silicone in a dose-dependent manner, with no effect of mineral oil. Release kinetics were then examined in vivo (Experiment 2) in nulliparous beef heifers assigned randomly to six groups (n = 6/group) and given either a large surface area (LSA) with 5% or 15% drug load, a standard surface area (SSA) intravaginal silicone device impregnated with 10% or 15% drug load, a wax dip-coat device (positive control), or a blank device (negative control). Devices were inserted on Day 3 (Day 0 = ovulation) until Day 11. Blood samples were collected at 0, 30 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, and twice daily until Day 11 to determine letrozole plasma concentrations by LC-MS/MS and estradiol plasma concentrations by radioimmunoassay The ovaries were examined once daily from Day 3-13 by ultrasonography to determine follicular and luteal responses to treatment. Letrozole plasma concentrations were higher in heifers given a device with an LSA vs SSA (P < 0.05). Plasma concentrations of estradiol decreased the most in heifers given the 15% LSA device (P = 0.06). The interval between emergence of successive follicular waves was longest (P < 0.05) in the positive control and the 15% LSA groups. As well, the diameter profiles of the dominant follicle and the corpus luteum were largest (P < 0.01) in the positive control and 15% LSA groups. In conclusion, letrozole was released from a silicone matrix in vitro in a dose-dependent manner, and the 15% LSA devices achieved target effects on ovarian function. Results may be used to manufacture a silicone intravaginal device for delivering aromatase inhibitors in a novel synchronization protocol for cattle.

Artificial insemination in South American camelids and wild equids.

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the "stallion effect", extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.

Banking North American buffalo semen.

The purpose of this study was to develop a procedure to collect and preserve semen from wood bison (Bison bison athabascae) and plains bison (Bison bison bison). Semen samples from three wood and three plains bison bulls were collected by electroejaculation from June through October. In addition, sperm was collected from the cauda epididymis of seven plains bison. Semen was cryopreserved using two commercially available cryopreservation media, an egg yolk-based medium (Triladyl), and a medium free of products of animal origin (Andromed). Sperm morphology and motility were recorded on fresh and post-thawed semen samples. Total sperm motility was not different between plains and wood bison for the months of June (50%), July (69%) and October (54%). However, total sperm motility for wood bison was higher (P<0.05) than plains bison for the months of August and September (August: 80% vs 55%; September: 73% vs 40%). Plains and wood bison did not differ in mean total and mean progressive motility (35 and 15%, respectively) of frozen-thawed sperm samples. The post-thaw motility of Triladyl-treated sperm was higher (P<0.05) than Andromed-treated sperm (35% vs 13%, respectively). Interestingly, post-thawed epididymal spermatozoa had higher total motility (P<0.05) than post-thawed electroejaculated sperm when cryopreserved with a medium free of products of animal origin (Andromed; 35% vs 9%, respectively). In conclusion, we used electroejaculation to collect high quality bison semen, and cryopreserved it for future needs.

Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Induction of ovarian follicular wave emergence in wapiti (Cervus elaphus).

Two experiments were done to test the effects of treatments designed to electively induce ovarian follicular wave emergence in wapiti for the purpose of group synchronization. In Experiment 1, hinds were assigned randomly to three groups and given saline im (controls; n=5), 5mg of estadiol-17beta im (n=4), or 5mg estradiol-17beta plus 100mg progesterone im (n=5). In Experiment 2, hinds were assigned randomly to two groups and given no treatment (controls; n=6), or transvaginal ultrasound-guided follicle ablation (n=7). In both experiments, ovarian follicular dynamics were monitored by daily transrectal ultrasonography from Day 0 (day of treatment) to Day 9. In Experiment 1, blood samples were collected at each examination for measurement of serum concentrations of progesterone and FSH. Both experiments were conducted during the late anestrous period (July and August). The mean (+/-S.E.M.) day of wave emergence did not differ between the control and estradiol alone groups, but tended to be later in the estradiol plus progesterone group Day 4.0+/-0.7, Day 3.5+/-0.3, and Day 5.2+/-0.2, respectively; P=0.06). The interval from treatment to wave emergence was less variable in the estradiol plus progesterone group (P<0.05) and tended to be less variable in the estradiol-alone group (P=0.07) than in the control group. The day of wave emergence was more variable (P<0.05) and tended to be later (P=0.10) in the control group compared to the ablation group (Day 2.5+/-0.8 versus Day 1.4+/-0.2). All three treatments were effective in synchronizing ovarian follicular wave emergence among a group of wapiti hinds. Follicle ablation may be an alternative method for synchronization of follicular waves in estrus synchronization and superstimulatory protocols.

Alternative approaches to setting up donor cows for superstimulation.

Protocols that controlled follicular wave emergence and ovulation have had a great impact on the application of on-farm embryo transfer, as they permitted the initiation of superstimulatory treatments at a self-appointed time. However, the most commonly used approach for synchronization of follicular wave emergence involved estradiol, which cannot be used in many countries. Therefore, alternative treatments are required. Mechanical removal of the dominant follicle by ultrasound-guided follicle aspiration was effective, but required the use of specialized equipment and trained technical staff, which made it difficult to utilize in the field. Exogenous GnRH or pLH have also been used to induce ovulation of a dominant follicle, synchronizing follicular wave emergence, but their efficacy was dependent on the stage of the dominant follicle at treatment; thus, the emergence of the ensuing follicular wave may be too variable for superstimulation. An alternative approach could be initiating treatments at the time of emergence of the first follicular wave, but the need to synchronize ovulation may be a disadvantage in groups of donors at random stages of the estrous cycle. The final alternative may be to use FSH or eCG to initiate a new wave, without regard to the presence of a dominant follicle, followed by superstimulatory treatment at a predetermined time. All alternatives need to be thoroughly investigated in order to confirm their utility in the superstimulation of donor cows, regardless of the stage of the estrous cycle and without compromising ova/embryo production.